This invention relates to ribozymes.
The following is a brief description of publications concerning ribozymes. None are admitted to be the prior art to the pending claims, and all are incorporated by reference herein.
The self-cleaving RNAs found in viruses and virusoids constitute a class of RNA catalysts, and their potential interactions with proteins in vivo remains largely unknown. These ribozymes can be converted into trans acting ribozymes that catalyze the site specific cleavage of an RNA substrate molecule (Hampel & Tritz, 1989 Biochemistry 28, 4929; Perrotta & Been, 1992 Biochemistry 31, 16; Uhlenbeck, 1987 Nature 328, 596).
Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Table I summarizes some of the characteristics of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
The specificity of the ribozyme can be altered in a predictable manner without loss of catalytic efficiency, and for this reason, such ribozymes have considerable potential for inactivating gene expression through the cleavage of targeted RNA (Cech, 1988 JAMA 260, 3030). In vivo, a targeted ribozyme may encounter many obstacles, including the ability to find its cognate substrate, fold into a catalytically active conformation, resist cellular nucleases, be able to discriminate its target among other RNAs, and eventually turn over to repeat the process.
The enzymatic nature of a ribozyme is advantageous over other technologies, such as antisense technology (where a nucleic acid molecule generally simply binds to a nucleic acid target to block its translation) since the concentration of ribozyme necessary to affect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme (Chowrira & Burke, 1991 Biochemistry 30, 8518; Joseph et al., 1993 Genes & Develop. 7, 30). Similar mismatches in antisense molecules do not prevent their action (Woolf et al., 1992 Proc. Natl. Acad. Sci. USA, 89, 7305-7309). Thus, the specificity of action of a ribozyme is greater than that of an antisense oligonucleotide binding the same RNA site.
The following publications generally discuss ribozymes, and in particular hairpin ribozymes. Van Tol et al., 1991 (Virology 180, 23) describe a hairpin ribozyme structure able to circularize. Hisamatsu et al., 1993 (Nucleic Acids Symp. Ser. 29, 173) describe hairpin ribozymes having a long substrate binding site in helix 1. Berzal-Herranz et al., 1993 (EMBO J. 12,2567) describe essential nucleotides in the hairpin ribozyme. Hampel and Tritz, 1989 (Biochemistry 28, 4929) describe a hairpin ribozyme derived from the minus strand of tobacco ringspot virus satellite [(-) sTRSV] RNA. Haseloff and Gerlach 1989 (Gene 82, 43) describe sequences required for self-cleavage reactions catalyzed by the (-) sTRSV RNA. Feldstein et al., 1989 (Gene 82, 53) tested various models of transcleaving motifs derived from (-) sTRSV RNAs. The hairpin ribozyme can be assembled in various combinations to catalyze a unimolecular, bimolecular or a trimolecular cleavage/ligation reaction (Berzal-Herranz et al., 1992, Genes & Develop. 6, 129; Chowrira and Burke, 1992 Nucleic Acids Res. 20, 2835; Komatsu et al., 1993 Nucleic Acids Res. 21, 185; Komatsu et al., 1994 J. Am. Chem. Soc. 116, 3692; Chowrira et al., 1994 J. Biol. Chem. 269, 25856). Increasing the length of helix 1 and helix 4 regions do not significantly affect the catalytic activity of the hairpin ribozyme (Hisamatsu et al., 1993 supra; Chowrira and Burke, 1992 supra; Anderson et al., 1994 Nucleic Acids Res. 22, 1096). For a review of various ribozyme motifs, and hairpin ribozyme in particular, see Burke, 1994 Nucleic Acids & Mol. Biol. 8, 105, eds. Eckstein and Lilley, Springer-Verlag, Germany; Ahsen and Schroeder, 1993 Bioessays 15, 299; Cech, 1992 Curr. Opi. Struc. Bio. 2, 605; and Hampel et al., 1993 Methods: A Companion to Methods in Enzymology 5, 37.
A hairpin ribozyme.circle-solid.substrate complex includes two intermolecular helices formed between the ribozyme and the target RNA (helix 1 and helix 2). The length of helix 1 can be varied substantially without affecting the catalytic activity of the ribozyme (Hisamatsu et al., 1993 supra). However, the length of helix 2 is reported to be sensitive to variation. The length of helix 2 is normally between 3 and 5 base-pairs long (Hampel & Tritz, 1989 supra; Feldstein et al. 1989 supra; Haseloff and Gerlach, 1989 supra, Hampel et al., 1990 supra; Feldstein et al., 1990 Proc. Natl. Acad. Sci. USA 87, 2623). Several reports suggest that mutations within this helix significantly inhibit ribozyme activity (Hampel et al., 1990 supra; Feldstein et al., 1990 supra; Chowrira & Burke, 1991 Biochemistry 30, 8518; Joseph et al., 1993 Genes & Develop. 7, 130). It is also believed in the art that the length of helix 2 should be between 3 and 5 bp (Hampel et al., 1988 EPO 360 257; Hampel et al., 1993 supra, Cech, 1992 supra; von Ahsen and Schroeder, 1993 supra; Hisamatsu et al., 1993 supra, Anderson et al., 1994 supra).
RNA-protein interactions are involved in many fundamental cellular processes. Well known examples of such ribonucleoproteins include RNase P, telomerase, Signal Recognition particles (SRP), the spliceosome, and the ribosome. Other examples of RNA-protein interactions occur during RNA processing and polyadenylation, and RNA trafficking within the cell.
It has been clearly demonstrated in the case of prokaryotic RNAse P and group I and II introns that the RNA is the catalytic component of the reaction. There is also accumulating evidence that 23S RNA of the ribosome (Noller et al., 1992 Science 256, 1416), and the snRNP RNAs (Guthrie, 1991 Science 253, 157) can at least partially catalyze translation and splicing, respectively. For the two latter examples, there is obviously an absolute requirement for many protein factors. At least 50 different proteins are involved in spliceosome assembly and function, up to 82 proteins in the ribosome, with many more required for translation. In the group I and group II introns, splicing is improved (in velocity and accuracy) by protein splicing factors (Coetze et al., 1994 Genes & Develop. 8, 1575; Mohr et al., 1994 Nature 370, 147; Saldanha et al., 1993 FASEB. J. 7, 15). Similarly, the protein component of RNase P is required for activity in vivo, and facilitates pre-tRNA cleavage in vitro (Altman et al., 1993 FASEB. J 7, 7). Thus, even if RNA is an efficient catalyst in vitro, in vivo RNA catalysis occurs in concert with proteins. Another important difference between in vitro and in vivo RNA catalysis is that RNA molecules are not homogeneously diffused throughout the cellular milieu. RNAs are dispatched in the cell by a still unknown protein machinery, and RNA-protein interactions are a key step in this mechanism (Wilhelm & Vale, 1993 J. Cell. Biol. 123, 269).
Jennings et al., U.S. Pat. No. 5,298,612 describe potential hammerhead ribozyme.circle-solid.protein interactions. It states:
Applicants have found that base-pairing in the group P is not required for cleavage of a target RNA. Accordingly, when nucleotide sequences X and Y are comprised solely of ribonucleotides and the group P is comprised solely of ribonucleotides, the ribonucleotides of group P may be base-paired for purpose other than to effect cleavage of a target RNA. Such purposes would include to allow the binding of cellular factors, such as RNA binding proteins or other cellular factors. Similarly, where the nucleotide sequences X and Y are comprised solely of deoxyribonucleotides, and the group P is comprised solely of deoxyribonucleotides, the deoxyribonucleotides of the group P may be base-paired for purposes other than involvement in endonuclease cleavage, such as interaction with DNA binding proteins or other cellular factors, which may, for example, effect cellular distribution of the endonuclease.